Cold-adapted equine influenza viruses

ABSTRACT

The present invention provides experimentally-generated cold-adapted equine influenza viruses, and reassortant influenza A viruses comprising at least one genome segment of such an equine influenza virus, wherein the equine influenza virus genome segment confers at least one identifying phenotype of the cold-adapted equine influenza virus, such as cold-adaptation, temperature sensitivity, dominant interference, or attenuation. Such viruses are formulated into therapeutic compositions to protect animals from diseases caused by influenza A viruses, and in particular, to protect horses from disease caused by equine influenza virus. The present invention also includes methods to protect animals from diseases caused by influenza A virus utilizing the claimed therapeutic compositions. Such methods include using a therapeutic composition as a vaccine to generate a protective immune response in an animal prior to exposure to a virulent virus, and using a therapeutic composition as a treatment for an animal that has been recently infected with a virulent virus, or is likely to be subsequently exposed to virulent virus in a few days whereby the therapeutic composition interferes with the growth of the virulent virus, even in the absence of immunity. The present invention also provides methods to produce cold-adapted equine influenza viruses, and reassortant influenza A viruses having at least one genome segment of an equine influenza virus generated by cold-adaptation.

FIELD OF THE INVENTION

[0001] The present invention relates to experimentally-generatedcold-adapted equine influenza viruses, and particularly to cold-adaptedequine influenza viruses having additional phenotypes, such asattenuation, dominant interference, or temperature sensitivity. Theinvention also includes reassortant influenza A viruses which contain atleast one genome segment from such an equine influenza virus, such thatthe reassortant virus includes certain phenotypes of the donor equineinfluenza virus. The invention further includes genetically-engineeredequine influenza viruses, produced through reverse genetics, whichcomprise certain identifying phenotypes of a cold-adapted equineinfluenza virus of the present invention. The present invention alsorelates to the use of these viruses in therapeutic compositions toprotect animals from diseases caused by influenza viruses.

BACKGROUND OF THE INVENTION

[0002] Equine influenza virus has been recognized as a major respiratorypathogen in horses since about 1956. Disease symptoms caused by equineinfluenza virus can be severe, and are often followed by secondarybacterial infections. Two subtypes of equine influenza virus arerecognized, namely subtype-1, the prototype being A/Equine/Prague/1/56(H7N7), and subtype-2, the prototype being A/Equine/Miami/1/63 (H3N8).Presently, the predominant virus subtype is subtype-2, which has furtherdiverged among Eurasian and North American isolates in recent years.

[0003] The currently licensed vaccine for equine influenza is aninactivated (killed) virus vaccine. This vaccine provides minimal, ifany, protection for horses, and can produce undesirable side effects,for example, inflammatory reactions at the site of injection. See, e.g.,Mumford, 1987, Equine Infectious Disease IV, 207-217, and Mumford, etal., 1993, Vaccine 11, 1172-1174. Furthermore, current modalities cannotbe used in young foals, because they cannot overcome maternal immunity,and can induce tolerance in a younger animal. Based on the severity ofdisease, there remains a need for safe, effective therapeuticcompositions to protect horses against equine influenza disease.

[0004] Production of therapeutic compositions comprising cold-adaptedhuman influenza viruses is described, for example, in Maassab, et al.,1960, Nature 7,612-614, and Maassab, et al., 1969, J. Immunol. 102,728-732. Furthermore, these researchers noted that cold-adapted humaninfluenza viruses, i.e., viruses that have been adapted to grow at lowerthan normal temperatures, tend to have a phenotype wherein the virus istemperature sensitive; that is, the virus does not grow well at certainhigher, non-permissive temperatures at which the wild-type virus willgrow and replicate. Various cold-adapted human influenza A viruses,produced by reassortment with existing cold-adapted human influenza Aviruses, have been shown to elicit good immune responses in vaccinatedindividuals, and certain live attenuated cold-adapted reassortant humaninfluenza A viruses have proven to protect humans against challenge withwild-type virus. See, e.g., Clements, et al., 1986, J. Clin. Microbiol.23, 73-76. In U.S. Pat. No. 5,149,531, by Youngner, et al., issued Sep.22, 1992, the inventors of the present invention further demonstratedthat certain reassortant cold-adapted human influenza A viruses alsopossess a dominant interference phenotype, i.e., they inhibit the growthof their corresponding parental wild-type strain, as well asheterologous influenza A viruses.

[0005] U.S. Pat. No. 4,683,137, by Coggins et al., issued Jul. 28, 1987,and U.S. Pat. No. 4,693,893, by Campbell, issued Sep. 15, 1987, discloseattenuated therapeutic compositions produced by reassortment ofwild-type equine influenza viruses with attenuated, cold-adapted humaninfluenza A viruses. Although these therapeutic compositions appear tobe generally safe and effective in horses, they pose a significantdanger of introducing into the environment a virus containing both humanand equine influenza genes.

SUMMARY OF THE INVENTION

[0006] The present invention provides experimentally-generatedcold-adapted equine influenza viruses, reassortant influenza A virusesthat comprise at least one genome segment of an equine influenza virusgenerated by cold-adaptation such that the equine influenza virus genomesegment confers at least one identifying phenotype of a cold-adaptedequine influenza virus on the reassortant virus, andgenetically-engineered equine influenza viruses, produced throughreverse genetics, which comprise at least one identifying phenotype of acold-adapted equine influenza virus. Identifying phenotypes includecold-adaptation, temperature sensitivity, dominant interference, andattenuation. The invention further provides a therapeutic composition toprotect an animal against disease caused by an influenza A virus, wherethe therapeutic composition includes a cold-adapted equine influenzavirus a reassortant influenza A virus, or a genetically-engineeredequine influenza virus of the present invention. Also provided is amethod to protect an animal from diseases caused by an influenza A viruswhich includes the administration of such a therapeutic composition.Also provided are methods to produce a cold-adapted equine influenzavirus, and methods to produce a reassortant influenza A virus whichcomprises at least one genome segment of a cold-adapted equine influenzavirus, where the equine influenza genome segment confers on thereassortant virus at least one identifying phenotype of the cold-adaptedequine influenza virus.

[0007] A cold-adapted equine influenza virus is one that replicates inembryonated chicken eggs at a temperature ranging from about 26° C. toabout 30° C. Preferably, a cold-adapted equine influenza virus,reassortant influenza A virus, or genetically-engineered equineinfluenza virus of the present invention is attenuated, such that itwill not cause disease in a healthy animal.

[0008] In one embodiment, a cold-adapted equine influenza virus,reassortant influenza A virus, or genetically-engineered equineinfluenza virus of the present invention is also temperature sensitive,such that the virus replicates in embryonated chicken eggs at atemperature ranging from about 26° C. to about 30° C., forms plaques intissue culture cells at a permissive temperature of about 34° C., butdoes not form plaques in tissue culture cells at a non-permissivetemperature of about 39° C.

[0009] In one embodiment, such a temperature sensitive virus comprisestwo mutations: a first mutation that inhibits plaque formation at atemperature of about 39° C., that mutation co-segregating with thegenome segment that encodes the viral nucleoprotein gene; and a secondmutation that inhibits all viral protein synthesis at a temperature ofabout 39° C.

[0010] In another embodiment, a cold-adapted, temperature sensitiveequine influenza virus of the present invention replicates inembryonated chicken eggs at a temperature ranging from about 26° C. toabout 30° C., forms plaques in tissue culture cells at a permissivetemperature of about 34° C., but does not form plaques in tissue culturecells or express late viral proteins at a non-permissive temperature ofabout 37° C.

[0011] Typically, a cold-adapted equine influenza virus of the presentinvention is produced by passaging a wild-type equine influenza virusone or more times, and then selecting viruses that stably grow andreplicate at a reduced temperature. A cold-adapted equine influenzavirus produced thereby includes, in certain embodiments, a dominantinterference phenotype, that is, the virus, when co-infected with aparental equine influenza virus or heterologous wild-type influenza Avirus, will inhibit the growth of that virus.

[0012] Examples of cold-adapted equine influenza viruses of the presentinvention include EIV-P821, identified by accession No. ATCC VR ______;EIV-P824, identified by accession No. ATCC VR ______; EIV-MSV+5,identified by accession No. ATCC VR ______; and progeny of such viruses.

[0013] Therapeutic compositions of the present invention include fromabout 10⁵ TCID₅₀ units to about 10⁸ TCID₅₀ units, and preferably about2×10⁶ TCID₅₀ units, of a cold-adapted equine influenza virus,reassortant influenza A virus, or genetically-engineered equineinfluenza virus of the present invention.

[0014] The present invention also includes a method to protect an animalfrom disease caused by an influenza A virus, which includes the step ofadministering to the animal a therapeutic composition including acold-adapted equine influenza virus, a reassortant influenza A virus, ora genetically-engineered equine influenza virus of the presentinvention. Preferred animals to protect include equids, with horses andponies being particularly preferred.

[0015] Yet another embodiment of the present invention is a method togenerate a cold-adapted equine influenza virus. The method includes thesteps of passaging a wild-type equine influenza virus; and selectingviruses that grow at a reduced temperature. In one embodiment, themethod includes repeating the passaging and selection steps one or moretimes, while progressively reducing the temperature. Passaging of equineinfluenza virus preferably takes place in embryonated chicken eggs.

[0016] Another embodiment is an method to produce a reassortantinfluenza A virus through genetic reassortment of the genome segments ofa donor cold-adapted equine influenza virus of the present inventionwith the genome segments of a recipient influenza A virus. Reassortantinfluenza A viruses of the present invention are produced by a methodthat includes the steps of: (a) mixing the genome segments of a donorcold-adapted equine influenza virus with the genome segments of arecipient influenza A virus, and (b) selecting viruses which include atleast one identifying phenotype of the donor equine influenza virus.Identifying phenotypes include cold-adaptation, temperature sensitivity,dominant interference, and attenuation. Preferably, such reassortantviruses at least include the attenuation phenotype of the donor virus. Atypical reassortant virus will have the antigenicity of the recipientvirus, that is, it will retain the hemagglutinin (HA) and neuraminidase(NA) phenotypes of the recipient virus.

[0017] The present invention further provides methods to propagatecold-adapted equine influenza viruses or reassortant influenza A virusesof the present invention. These methods include propagation inembryonated chicken eggs or in tissue culture cells.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention provides experimentally-generatedcold-adapted equine influenza viruses comprising certain definedphenotypes, which are disclosed herein. It is to be noted that the term“a” or “an” entity, refers to one or more of that entity; for example,“a cold-adapted equine influenza virus” can include one or morecold-adapted equine influenza viruses. As such, the terms “a” (or “an”),“one or more,” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising,” “including,” and“having” can be used interchangeably. Furthermore, an item “selectedfrom the group consisting of” refers to one or more of the items in thatgroup, including combinations thereof.

[0019] A cold-adapted equine influenza virus of the present invention isa virus that has been generated in the laboratory, and as such, is not avirus as occurs in nature. Since the present invention also includesthose viruses having the identifying phenotypes of such a cold-adaptedequine influenza virus, an equine influenza virus isolated from amixture of naturally-occurring viruses, i.e., removed from its naturalmilieu, but having the claimed phenotypes, is included in the presentinvention. A cold-adapted equine influenza virus of the presentinvention does not require any specific level of purity. For example, acold-adapted equine influenza virus grown in embryonated chicken eggsmay be in a mixture with the allantoic fluid (AF), and a cold-adaptedequine influenza virus grown in tissue culture cells may be in a mixturewith disrupted cells and tissue culture medium.

[0020] As used herein, an “equine influenza virus” is an influenza virusthat infects and grows in equids, e.g., horses or ponies. As usedherein, “growth” of a virus denotes the ability of the virus toreproduce or “replicate” itself in a permissive host cell. As such, theterms, “growth of a virus” and “replication of a virus” are usedinterchangeably herein. Growth or replication of a virus in a particularhost cell can be demonstrated and measured by standard methodswell-known to those skilled in the art of virology. For example, samplescontaining infectious virus, e.g., as contained in nasopharyngealsecretions from an infected horse, are tested for their ability to causecytopathic effect (CPE), e.g., virus plaques, in tissue culture cells.Infectious virus may also be detected by inoculation of a sample intothe allantoic cavity of embryonated chicken eggs, and then testing theAF of eggs thus inoculated for its ability to agglutinate red bloodcells, i.e., cause hemagglutination, due to the presence of theinfluenza virus hemagglutinin (HA) protein in the AF.

[0021] Naturally-occurring, i.e., wild-type, equine influenza virusesreplicate well at a temperature from about 34° C. to about 39° C. Forexample, wild-type equine influenza virus replicates in embryonatedchicken eggs at a temperature of about 34° C., and replicates in tissueculture cells at a temperature from about 34° C. to about 39° C. As usedherein, a “cold-adapted” equine influenza virus is an equine influenzavirus that has been adapted to grow at a temperature lower than theoptimal growth temperature for equine influenza virus. One example of acold-adapted equine influenza virus of the present invention is a virusthat replicates in embryonated chicken eggs at a temperature of about30° C. A preferred cold-adapted equine influenza virus of the presentinvention replicates in embryonated chicken eggs at a temperature ofabout 28° C. Another preferred cold-adapted equine influenza virus ofthe present invention replicates in embryonated chicken eggs at atemperature of about 26° C. In general, preferred cold-adapted equineinfluenza viruses of the present invention replicate in embryonatedchicken eggs at a temperature ranging from about 26° C. to about 30° C.,i.e., at a range of temperatures at which a wild-type virus will growpoorly or not at all. It should be noted that the ability of suchviruses to replicate within that temperature range does not precludetheir ability to also replicate at higher or lower temperatures. Forexample, one embodiment is a cold-adapted equine influenza virus thatreplicates in embryonated chicken eggs at a temperature of about 26° C.,but also replicates in tissue culture cells at a temperature of about34° C. As with wild-type equine influenza viruses, cold-adapted equineinfluenza viruses of the present invention generally form plaques intissue culture cells, for example Madin Darby Canine Kidney Cells (MDCK)at a temperature of about 34° C. Examples of suitable and preferredcold-adapted equine influenza viruses of the present invention aredisclosed herein.

[0022] One embodiment of the present invention is a cold-adapted equineinfluenza virus that is produced by a method which includes passaging awild-type equine influenza virus, and then selecting viruses that growat a reduced temperature. Cold-adapted equine influenza viruses of thepresent invention can be produced, for example, by sequentiallypassaging a wild-type equine influenza virus in embryonated chicken eggsat progressively lower temperatures, thereby selecting for certainmembers of the virus mixture which stably replicate at the reducedtemperature. An example of a passaging procedure is disclosed in detailin the Examples section. During the passaging procedure, one or moremutations appear in certain of the single-stranded RNA segmentscomprising the influenza virus genome, which alter the genotype, i.e.,the primary nucleotide sequence of those RNA segments. As used herein, a“mutation” is an alteration of the primary nucleotide sequence of anygiven RNA segment making up an influenza virus genome. Examples ofmutations include substitution of one or more nucleotides, deletion ofone or more nucleotides, insertion of one or more nucleotides, orinversion of a stretch of two or more nucleotides. By selecting forthose members of the virus mixture that stably replicate at a reducedtemperature, a virus with a cold-adaptation phenotype is selected. Asused herein, a “phenotype” is an observable or measurable characteristicof a biological entity such as a cell or a virus, where the observedcharacteristic is attributable to a specific genetic configuration ofthat biological entity, i.e., a certain genotype. As such, acold-adaptation phenotype is the result of one or more mutations in thevirus genome. As used herein, the terms “a mutation,” “a genome,” “agenotype,” or “a phenotype”refer to one or more, or at least onemutation, genome, genotype, or phenotype, respectively.

[0023] Additional, observable phenotypes in a cold-adapted equineinfluenza virus may occur, and will generally be the result of one ormore additional mutations in the genome of such a virus. For example, acold-adapted equine influenza virus of the present invention may, inaddition, be attenuated, exhibit dominant interference, and/or betemperature sensitive.

[0024] In one embodiment, a cold-adapted equine influenza virus of thepresent invention has a phenotype characterized by attenuation. Acold-adapted equine influenza virus is “attenuated,” when administrationof the virus to an equine influenza virus-susceptible animal results inreduced or absent clinical signs in that animal, compared to clinicalsigns observed in animals that are infected with wild-type equineinfluenza virus. For example, an animal infected with wild-type equineinfluenza virus will display fever, sneezing, coughing, depression, andnasal discharges. In contrast, an animal administered an attenuated,cold-adapted equine influenza virus of the present invention willdisplay minimal or no, i.e., undetectable, clinical disease signs.

[0025] In another embodiment, a cold-adapted equine influenza virus ofthe present invention comprises a temperature sensitive phenotype. Asused herein, a temperature sensitive cold-adapted equine influenza virusreplicates at reduced temperatures, but no longer replicates or formsplaques in tissue culture cells at certain higher growth temperatures atwhich the wild-type virus will replicate and form plaques. While notbeing bound by theory, it is believed that replication of equineinfluenza viruses with a temperature sensitive phenotype is largelyrestricted to the cool passages of the upper respiratory tract, and doesnot replicate efficiently in the lower respiratory tract, where thevirus is more prone to cause disease symptoms. A temperature at which atemperature sensitive virus will grow is referred to herein as a“permissive” temperature for that temperature sensitive virus, and ahigher temperature at which the temperature sensitive virus will notgrow, but at which a corresponding wild-type virus will grow, isreferred to herein as a “non-permissive” temperature for thattemperature sensitive virus. For example, certain temperature sensitivecold-adapted equine influenza viruses of the present invention replicatein embryonated chicken eggs at a temperature at or below about 30° C.,preferably at about 28° C. or about 26° C., and will form plaques intissue culture cells at a permissive temperature of about 34° C., butwill not form plaques in tissue culture cells at a non-permissivetemperature of about 39° C. Other temperature sensitive cold-adaptedequine influenza viruses of the present invention replicate inembryonated chicken eggs at a temperature at or below about 30° C.,preferably at about 28° C. or about 26° C., and will form plaques intissue culture cells at a permissive temperature of about 34° C., butwill not form plaques in tissue culture cells at a non-permissivetemperature of about 37° C.

[0026] Certain cold-adapted equine influenza viruses of the presentinvention have a dominant interference phenotype; that is, they dominatean infection when co-infected into cells with another influenza A virus,thereby impairing the growth of that other virus. For example, when acold-adapted equine influenza virus of the present invention, having adominant interference phenotype, is co-infected into MDCK cells with thewild-type parental equine influenza virus, A/equine/Kentucky/1/91(H3N8), growth of the parental virus is impaired. Thus, in an animalthat has recently been exposed to, or may be soon exposed to, a virulentinfluenza virus, i.e., an influenza virus that causes disease symptoms,administration of a therapeutic composition comprising a cold-adaptedequine influenza virus having a dominant interference phenotype into theupper respiratory tract of that animal will impair the growth of thevirulent virus, thereby ameliorating or reducing disease in that animal,even in the absence of an immune response to the virulent virus.

[0027] Dominant interference of a cold-adapted equine influenza virushaving a temperature sensitive phenotype can be measured by standardvirological methods. For example, separate monolayers of MDCK cells canbe infected with (a) a virulent wild-type influenza A virus, (b) atemperature sensitive, cold-adapted equine influenza virus, and (c) bothviruses in a co-infection, with all infections done at multiplicities ofinfection (MOI) of about 2 plaque forming units (pfu) per cell. Afterinfection, the virus yields from the various infected cells are measuredby duplicate plaque assays performed at the permissive temperature forthe cold-adapted equine influenza virus and at the non-permissivetemperature of that virus. A cold adapted equine influenza virus havinga temperature sensitive phenotype is unable to form plaques at itsnon-permissive temperature, while the wild-type virus is able to formplaques at both the permissive and non-permissive temperatures. Thus itis possible to measure the growth of the wild-type virus in the presenceof the cold adapted virus by comparing the virus yield at thenon-permissive temperature of the cells singly infected with wild-typevirus to the yield at the non-permissive temperature of the wild-typevirus in doubly infected cells.

[0028] Cold-adapted equine influenza viruses of the present inventionare characterized primarily by one or more of the following identifyingphenotypes: cold-adaptation, temperature sensitivity, dominantinterference, and/or attenuation. As used herein, the phrase “an equineinfluenza virus comprises the identifying phenotype(s) ofcold-adaptation, temperature sensitivity, dominant interference, and/orattenuation” refers to a virus having such a phenotype(s). Examples ofsuch viruses include, but are not limited to, EIV-P821, identified byaccession No. ATCC VR ______, EIV-P824, identified by accession No. ATCCVR ______, and EIV-MSV+5, identified by accession No. ATCC VR ______, aswell as EIV-MSVO, EIV, MSV+1, EIV-MSV+2, EIV-MSV+3, and EIV-MSV+4.Production of such viruses is described in the examples. For example,cold-adapted equine influenza virus EIV-P821 is characterized by, i.e.,has the identifying phenotypes of, (a) cold-adaptation, e.g., itsability to replicate in embryonated chicken eggs at a temperature ofabout 26° C.; (b) temperature sensitivity, e.g., its inability to formplaques in tissue culture cells and to express late gene products at anon-permissive temperature of about 37° C., and its inability to formplaques in tissue culture cells and to synthesize any viral proteins ata non-permissive temperature of about 39° C.; (c) its attenuation uponadministration to an equine influenza virus-susceptible animal; and (d)dominant interference, e.g., its ability, when co-infected into a cellwith a wild-type influenza A virus, to interfere with the growth of thatwild-type virus. Similarly, cold-adapted equine influenza virus EIV-P824is characterized by (a) cold adaptation, e.g., its ability to replicatein embryonated chicken eggs at a temperature of about 28° C.; (b)temperature sensitivity, e.g., its inability to form plaques in tissueculture cells at a non-permissive temperature of about 39° C.; and (c)dominant interference, e.g., its ability, when co-infected into a cellwith a wild-type influenza A virus, to interfere with the growth of thatwild-type virus. In another example, cold-adapted equine influenza virusEIV-MSV+5 is characterized by (a) cold-adaptation, e.g., its ability toreplicate in embryonated chicken eggs at a temperature of about 26° C.;(b) temperature sensitivity, e.g., its inability to form plaques intissue culture cells at a non-permissive temperature of about 39° C.;and (c) its attenuation upon administration to an equine influenzavirus-susceptible animal.

[0029] In certain cases, the RNA segment upon which one or moremutations associated with a certain phenotype occur may be determinedthrough reassortment analysis by standard methods, as disclosed herein.In one embodiment, a cold-adapted equine influenza virus of the presentinvention comprises a temperature sensitive phenotype that correlateswith at least two mutations in the genome of that virus. In thisembodiment, one of the two mutations, localized by reassortment analysisas disclosed herein, inhibits, i.e., blocks or prevents, the ability ofthe virus to form plaques in tissue culture cells at a non-permissivetemperature of about 39° C. This mutation co-segregates with the segmentof the equine influenza virus genome that encodes the nucleoprotein (NP)gene of the virus, i.e., the mutation is located on the same RNA segmentas the NP gene. In this embodiment, the second mutation inhibits allprotein synthesis at a non-permissive temperature of about 39° C. Assuch, at the non-permissive temperature, the virus genome is incapableof expressing any viral proteins. Examples of cold-adapted equineinfluenza viruses possessing these characteristics are EIV-P821 and EIVMSV+5. EIV-P821 was generated by serial passaging of a wild-type equineinfluenza virus in embryonated chicken eggs by methods described inExample 1A. EIV-MSV+5 was derived by further serial passaging of EIV-P821, as described in Example 1E.

[0030] Furthermore, a cold-adapted, temperature sensitive equineinfluenza virus comprising the two mutations which inhibit plaqueformation and viral protein synthesis at a non-permissive temperature ofabout 39° C. can comprise one or more additional mutations, whichinhibit the virus' ability to synthesize late gene products and to formplaques in tissue culture cells at a non-permissive temperature of about37° C. An example of a cold-adapted equine influenza virus possessingthese characteristics is EIV-P821. This virus isolate replicates inembryonated chicken eggs at a temperature of about 26° C., and does notform plaques or express any viral proteins at a temperature of about 39°C. Furthermore, EIV-P821 does not form plaques on MDCK cells at anon-permissive temperature of about 37° C., and at this temperature,late gene expression is inhibited in such a way that late proteins arenot produced, i.e., normal levels of NP protein are synthesized, reducedor undetectable levels of M1 or HA proteins are synthesized, andenhanced levels of the polymerase proteins are synthesized. Since thisphenotype is typified by differential viral protein synthesis, it isdistinct from the protein synthesis phenotype seen at a non-permissivetemperature of about 39° C., which is typified by the inhibition ofsynthesis of all viral proteins.

[0031] Pursuant to 37 CFR § 1.802 (a-c), cold-adapted equine influenzaviruses, designated herein as EIV-P821, an EIV-P824 were deposited withthe American Type Culture Collection (ATCC, 10801 University Boulevard,Manassas, Va. 20110-2209) under the Budapest Treaty as ATCC AccessionNos. ATCC VR-______, and ATCC VR-______, respectively, on Jul. 11, 1998.Cold-adapted equine influenza virus EIV-MSV+5 was deposited with theATCC as ATCC Accession No. ATCC VR-______ on Aug. 3, 1998. Pursuant to37 CFR § 1.806, the deposits are made for a term of at least thirty (30)years and at least five (5) years after the most recent request for thefurnishing of a sample of the deposit was received by the depository.Pursuant to 37 CFR § 1.808 (a)(2), all restrictions imposed by thedepositor on the availability to the public will be irrevocably removedupon the granting of the patent.

[0032] Preferred cold-adapted equine influenza viruses of the presentinvention have the identifying phenotypes of EIV-P821, EIV-P824, andEIV-MSV+5. Particularly preferred cold-adapted equine influenza virusesinclude EIV-P821, EIV-P824, EIV-MSV+5, and progeny of these viruses. Asused herein, “progeny” are “offspring,” and as such can slightly alteredphenotypes compared to the parent virus, but retain identifyingphenotypes of the parent virus, for example, cold-adaptation,temperature sensitivity, dominant interference, or attenuation. Forexample, cold-adapted equine influenza virus EIV-MSV+5 is a “progeny” ofcold-adapted equine influenza virus EIV-P821. “Progeny” also includereassortant influenza A viruses that comprise one or more identifyingphenotypes of the donor parent virus.

[0033] Reassortant influenza A viruses of the present invention areproduced by genetic reassortment of the genome segments of a donorcold-adapted equine influenza virus of the present invention with thegenome segments of a recipient influenza A virus, and then selecting areassortant virus that derives at least one of its eight RNA genomesegments from the donor virus, such that the reassortant virus acquiresat least one identifying phenotype of the donor cold-adapted equineinfluenza virus. Identifying phenotypes include cold-adaptation,temperature sensitivity, attenuation, and dominant interference.Preferably, reassortant influenza A viruses of the present inventionderive at least the attenuation phenotype of the donor virus. Methods toisolate reassortant influenza viruses are well known to those skilled inthe art of virology and are disclosed, for example, in Fields, et al.,1996, Fields Virology, 3d ed, Lippincott-Raven; and Palese, et al.,1976, J. Virol., 17, 876-884. Fields, et al., ibid. and Palese, et al.,ibid. are incorporated herein by reference in their entireties.

[0034] A suitable donor equine influenza virus is a cold-adapted equineinfluenza virus of the present invention, for example, EIV-P821,identified by accession No. ATCC VR ______, EIV-P824, identified byaccession No. ATCC VR ______, or EIV-MSV+5, identified by accession No.ATCC VR ______. A suitable recipient influenza A virus can be anotherequine influenza virus, for example a Eurasian subtype 2 equineinfluenza virus such as A/equine/Suffolk/89 (H3N8) or a subtype 1 equineinfluenza virus such as A/Prague/1/56 (H7N7). A recipient influenza Avirus can also be any influenza A virus capable of forming a reassortantvirus with a donor cold-adapted equine influenza virus. Examples of suchinfluenza A viruses include, but are not limited to, human influenzaviruses such as A/Puerto Rico/8/34 (H1N1), A/Hong Kong/156/97 (H5N1),A/Singapore/1/57 (H2N2), and A/Hong Kong/1/68 (H3N2); swine viruses suchas A/Swine/Iowa/15/30 (H1N1); and avian viruses such as A/mallard/NewYork/6750/78 (H2N2) and A/chicken/Hong Kong/258/97 (H5N1). A reassortantvirus of the present invention can include any combination of donor andrecipient gene segments, as long as the resulting reassortant viruspossesses at least one identifying phenotype of the donor virus.

[0035] One example of a reassortant virus of the present invention is a“6+2” reassortant virus, in which the six “internal gene segments,”i.e., those comprising the NP, PB2, PB1, PA, M, and NS genes, arederived from the donor cold-adapted equine influenza virus genome, andthe two “external gene segments,” i.e., those comprising the HA and NAgenes, are derived from the recipient influenza A virus. A resultantvirus thus produced has the attenuated, cold-adapted, temperaturesensitive, and/or dominant interference phenotypes of the donorcold-adapted equine influenza virus, but the antigenicity of therecipient strain.

[0036] In yet another embodiment, a cold-adapted equine influenza virusof the present invention can be produced through recombinant means. Inthis approach, one or more specific mutations, associated withidentified cold-adaptation, attenuation, temperature sensitivity, ordominant interference phenotypes, are identified and are introduced backinto a wild-type equine influenza virus strain using a reverse geneticsapproach. Reverse genetics entails using RNA polymerase complexesisolated from influenza virus-infected cells to transcribe artificialinfluenza virus genome segments containing the mutation(s),incorporating the synthesized RNA segment(s) into virus particles usinga helper virus, and then selecting for viruses containing the desiredchanges. Reverse genetics methods for influenza viruses are described,for example, in Enami, et al., 1990, Proc. Natl. Acad. Sci. 87, 3802-3805; and in U.S. Pat. No. 5,578,473, by Palese, et al., issued Nov.26, 1996, both of which are incorporated herein by reference in theirentireties. This approach allows one skilled in the art to produceadditional cold-adapted equine influenza viruses of the presentinvention without the need to go through the lengthy cold-adaptationprocess, and the process of selecting mutants both in vitro and in vivowith the desired virus phenotype.

[0037] A cold-adapted equine influenza virus of the present inventionmay be propagated by standard virological methods well-known to thoseskilled in the art, examples of which are disclosed herein. For example,a cold-adapted equine influenza virus can be grown in embryonatedchicken eggs or in eukaryotic tissue culture cells. Suitable continuouseukaryotic cell lines upon which to grow a cold-adapted equine influenzavirus of the present invention include those that support growth ofinfluenza viruses, for example, MDCK cells. Other suitable cells uponwhich to grow a cold-adapted equine influenza virus of the presentinvention include, but are not limited to, primary kidney cell culturesof monkey, calf, hamster or chicken.

[0038] In one embodiment, the present invention provides a therapeuticcomposition to protect an animal against disease caused by an influenzaA virus, where the therapeutic composition includes either acold-adapted equine influenza virus or a reassortant influenza A viruscomprising at least one genome segment of an equine influenza virusgenerated by cold-adaptation, wherein the equine influenza virus genomesegment confers at least one identifying phenotype of the cold-adaptedequine influenza virus. In addition, a therapeutic composition of thepresent invention can include an equine influenza virus that has beengenetically engineered to comprise one or more mutations, where thosemutations have been identified to confer a certain identifying phenotypeon a cold-adapted equine influenza virus of the present invention. Asused herein, the phrase “disease caused by an influenza A virus” refersto the clinical manifestations observed in an animal which has beeninfected with a virulent influenza A virus. Examples of such clinicalmanifestations include, but are not limited to, fever, sneezing,coughing, nasal discharge, rales, anorexia and depression. In addition,the phrase “disease caused by an influenza A virus” is defined herein toinclude shedding of virulent virus by the infected animal. Verificationthat clinical manifestations observed in an animal correlate withinfection by virulent equine influenza virus may be made by severalmethods, including the detection of a specific antibody and/or T-cellresponses to equine influenza virus in the animal. Preferably,verification that clinical manifestations observed in an animalcorrelate with infection by a virulent influenza A virus is made by theisolation of the virus from the afflicted animal, for example, byswabbing the nasopharyngeal cavity of that animal for virus-containingsecretions. Verification of virus isolation may be made by the detectionof CPE in tissue culture cells inoculated with the isolated secretions,by inoculation of the isolated secretions into embryonated chicken eggs,where virus replication is detected by the ability of AF from theinoculated eggs to agglutinate erythrocytes, suggesting the presence ofthe influenza virus hemagglutinin protein, or by use of a commerciallyavailable diagnostic test, for example, the Directigen® FLU A test.

[0039] As used herein, the term “to protect” includes, for example, toprevent or to treat influenza A virus infection in the subject animal.As such, a therapeutic composition of the present invention can be used,for example, as a prophylactic vaccine to protect a subject animal frominfluenza disease by administering the therapeutic composition to thatanimal at some time prior to that animal's exposure to the virulentvirus.

[0040] A therapeutic composition of the present invention, comprising acold-adapted equine influenza virus having a dominant interferencephenotype, can also be used to treat an animal that has been recentlyinfected with virulent influenza A virus or is likely to be subsequentlyexposed in a few days, such that the therapeutic composition immediatelyinterferes with the growth of the virulent virus, prior to the animal'sproduction of antibodies to the virulent virus. A therapeuticcomposition comprising a cold-adapted equine influenza virus having adominant interference phenotype may be effectively administered prior tosubsequent exposure for a length of time corresponding to theapproximate length of time that a cold-adapted equine influenza virus ofthe present invention will replicate in the upper respiratory tract of atreated animal, for example, up to about seven days. A therapeuticcomposition comprising a cold-adapted equine influenza virus having adominant interference phenotype may be effectively administeredfollowing exposure to virulent equine influenza virus for a length oftime corresponding to the time required for an infected animal to showdisease symptoms, for example, up to about two days.

[0041] Therapeutic compositions of the present invention can beadministered to any animal susceptible to influenza virus disease, forexample, humans, swine, horses and other equids, aquatic birds, domesticand game fowl, seals, mink, and whales. Preferably, a therapeuticcomposition of the present invention is administered equids. Even morepreferably, a therapeutic composition of the present invention isadministered to a horse, to protect against equine influenza disease.

[0042] Current vaccines available to protect horses against equineinfluenza virus disease are not effective in protecting young foals,most likely because they cannot overcome the maternal antibody presentin these young animals, and often, vaccination at an early age, forexample 3 months of age, can lead to tolerance rather than immunity. Inone embodiment, and in contrast to existing equine influenza virusvaccines, a therapeutic composition comprising a cold-adapted equineinfluenza virus of the present invention apparently can produce immunityin young animals. As such, a therapeutic composition of the presentinvention can be safely and effectively administered to young foals, asyoung as about 3 months of age, to protect against equine influenzadisease without the induction of tolerance.

[0043] In one embodiment, a therapeutic composition of the presentinvention can be multivalent. For example, it can protect an animal frommore than one strain of influenza A virus by providing a combination ofone or more cold-adapted equine influenza viruses of the presentinvention, one or more reassortant influenza A viruses, and/or one ormore genetically-engineered equine influenza viruses of the presentinvention. Multivalent therapeutic compositions can include at least twocold-adapted equine influenza viruses, e.g., against North Americansubtype-2 virus isolates such as A/equine/Kentucky/1/91 (H1N8), andEurasian subtype-2 virus isolates such as A/equine/Suffolk/89 (H3N8); orone or more subtype-2 virus isolates and a subtype-1 virus isolate suchas A/equine/Prague/1/56 (H7N7). Similarly, a multivalent therapeuticcomposition of the present invention can include a cold-adapted equineinfluenza virus and a reassortant influenza A virus of the presentinvention, or two reassortant influenza A viruses of the presentinvention. A multivalent therapeutic composition of the presentinvention can also contain one or more formulations to protect againstone or more other infectious agents in addition to influenza A virus.Such other infectious agents include, but not limited to: viruses;bacteria; fungi and fungal-related microorganisms; and parasites.Preferable multivalent therapeutic compositions include, but are notlimited to, a cold-adapted equine influenza virus, reassortant influenzaA virus, or genetically-engineered equine influenza virus of the presentinvention plus one or more compositions protective against one or moreother infectious agents that afflict horses. Suitable infectious agentsto protect against include, but are not limited to, equine infectiousanemia virus, equine herpes virus, eastern, western, or Venezuelanequine encephalitis virus, tetanus, Streptococcus equi, and Ehrlichiaresticii.

[0044] A therapeutic composition of the present invention can beformulated in an excipient that the animal to be treated can tolerate.Examples of such excipients include water, saline, Ringer's solution,dextrose solution, Hank's solution, and other aqueous physiologicallybalanced salt solutions. Excipients can also contain minor amounts ofadditives, such as substances that enhance isotonicity and chemical orbiological stability. Examples of buffers include phosphate buffer,bicarbonate buffer, and Tris buffer, while examples of stabilizersinclude A1/A2 stabilizer, available from Diamond Animal Health, DesMoines, Iowa. Standard formulations can either be liquids or solidswhich can be taken up in a suitable liquid as a suspension or solutionfor administration to an animal. In one embodiment, a non-liquidformulation may comprise the excipient salts, buffers, stabilizers,etc., to which sterile water or saline can be added prior toadministration.

[0045] A therapeutic composition of the present invention may alsoinclude one or more adjuvants or carriers. Adjuvants are typicallysubstances that enhance the immune response of an animal to a specificantigen, and carriers include those compounds that increase thehalf-life of a therapeutic composition in the treated animal. Oneadvantage of a therapeutic composition comprising a cold-adapted equineinfluenza virus or a reassortant influenza A virus of the presentinvention is that adjuvants and carriers are not required to produce anefficacious vaccine. Furthermore, in many cases known to those skilledin the art, the advantages of a therapeutic composition of the presentinvention would be hindered by the use of some adjuvants or carriers.However, it should be noted that use of adjuvants or carriers is notprecluded by the present invention.

[0046] Therapeutic compositions of the present invention include anamount of a cold-adapted equine influenza virus that is sufficient toprotect an animal from challenge with virulent equine influenza virus.In one embodiment, a therapeutic composition of the present inventioncan include an amount of a cold-adapted equine influenza virus rangingfrom about 10⁵ tissue culture infectious dose-50 (TCID₅₀) units of virusto about 10⁸ TCID₅₀ units of virus. As used herein, a “TCID₅₀ unit” isamount of a virus which results in cytopathic effect in 50% of thosecell cultures infected. Methods to measure and calculate TCID₅₀ areknown to those skilled in the art and are available, for example, inReed and Muench, 1938, Am. J. of Hyg. 27, 493-497, which is incorporatedherein by reference in its entirety. A preferred therapeutic compositionof the present invention comprises from about 10⁶ TCID₅₀ units to about10⁷ TCID₅₀ units of a cold-adapted equine influenza virus or reassortantinfluenza A virus of the present invention. Even more preferred is atherapeutic composition comprising about 2×10⁶ TCID₅₀ units of acold-adapted equine influenza virus or reassortant influenza A virus ofthe present invention.

[0047] The present invention also includes methods to protect an animalagainst disease caused by an influenza A virus comprising administeringto the animal a therapeutic composition of the present invention.Preferred are those methods which protect an equid against diseasecaused by equine influenza virus, where those methods compriseadministering to the equid a cold-adapted equine influenza virus.Acceptable protocols to administer therapeutic compositions in aneffective manner include individual dose size, number of doses,frequency of dose administration, and mode of administration.Determination of such protocols can be accomplished by those skilled inthe art, and examples are disclosed herein.

[0048] A preferable method to protect an animal against disease causedby an influenza A virus includes administering to that animal a singledose of a therapeutic composition comprising a cold-adapted equineinfluenza virus, a reassortant influenza A virus, orgenetically-engineered equine influenza virus of the present invention.A suitable single dose is a dose that is capable of protecting an animalfrom disease when administered one or more times over a suitable timeperiod. The method of the present invention may also includeadministering subsequent, or booster doses of a therapeutic composition.Booster administrations can be given from about 2 weeks to several yearsafter the original administration. Booster administrations preferablyare administered when the immune response of the animal becomesinsufficient to protect the animal from disease. Examples of suitableand preferred dosage schedules are disclosed in the Examples section.

[0049] A therapeutic composition of the present invention can beadministered to an animal by a variety of means, such that the viruswill enter and replicate in the mucosal cells in the upper respiratorytract of the treated animal. Such means include, but are not limited to,intranasal administration, oral administration, and intraocularadministration. Since influenza viruses naturally infect the mucosa ofthe upper respiratory tract, a preferred method to administer atherapeutic composition of the present invention is by intranasaladministration. Such administration may be accomplished by use of asyringe fitted with cannula, or by use of a nebulizer fitted over thenose and mouth of the animal to be vaccinated.

[0050] The efficacy of a therapeutic composition of the presentinvention to protect an animal against disease caused by influenza Avirus can be tested in a variety of ways including, but not limited to,detection of antibodies by, for example, hemagglutination inhibition(HAI) tests, detection of cellular immunity within the treated animal,or challenge of the treated animal with virulent equine influenza virusto determine whether the treated animal is resistant to the developmentof disease. In addition, efficacy of a therapeutic composition of thepresent invention comprising a cold-adapted equine influenza virushaving a dominant interference phenotype to ameliorate or reduce diseasesymptoms in an animal previously inoculated or susceptible toinoculation with a virulent, wild-type equine influenza virus can betested by screening for the reduction or absence of disease symptoms inthe treated animal.

[0051] The present invention also includes methods to produce atherapeutic composition of the present invention. Suitable and preferredmethods for making a therapeutic composition of the present inventionare disclosed herein. Pertinent steps involved in producing one type oftherapeutic composition of the present invention, i.e., a cold-adaptedequine influenza virus, include (a) passaging a wild-type equineinfluenza virus in vitro, for example, in embryonated chicken eggs; (b)selecting viruses that grow at a reduced temperature; (c) repeating thepassaging and selection steps one or more times, at progressively lowertemperatures, until virus populations are selected which stably grow atthe desired lower temperature; and (d) mixing the resulting viruspreparation with suitable excipients.

[0052] The pertinent steps involved in producing another type oftherapeutic composition of the present invention, i.e., a reassortantinfluenza A virus having at least one genome segment of an equineinfluenza virus generated by adaptation, includes the steps of (a)mixing the genome segments of a donor cold-adapted equine influenzavirus, which preferably also has the phenotypes of attenuation,temperature sensitivity, or dominant interference, with the genomesegments of a recipient influenza A virus, and (b) selecting reassortantviruses that have at least one identifying phenotype of the donor equineinfluenza virus. Identifying phenotypes to select for includeattenuation, cold-adaptation, temperature sensitivity, and dominantinterference. Methods to screen for these phenotypes are well known tothose skilled in the art, and are disclosed herein. It is preferable toscreen for viruses that at least have the phenotype of attenuation.

[0053] Using this method to generate a reassortant influenza A virushaving at least one genome segment of a equine influenza virus generatedby cold-adaptation, one type of reassortant virus to select for is a“6+2” reassortant, where the six “internal gene segments,” i.e., thosecoding for the NP, PB2, PB1, PA, M, and NS genes, are derived from thedonor cold-adapted equine influenza virus genome, and the two “externalgene segments,” i.e., those coding for the HA and NA genes, are derivedfrom the recipient influenza A virus. A resultant virus thus producedcan have the cold-adapted, attenuated, temperature sensitive, and/orinterference phenotypes of the donor cold-adapted equine influenzavirus, but the antigenicity of the recipient strain.

[0054] The following examples are provided for the purposes ofillustration and are not intended to limit the scope of the presentinvention.

EXAMPLE 1

[0055] This example discloses the production and phenotypiccharacterization of several cold-adapted equine influenza viruses of thepresent invention.

[0056] A. Parental equine influenza virus, A/equine/Kentucky/1/91 (H3N8)(obtained from Tom Chambers, the University of Kentucky, Lexington, Ky.)was subjected to cold-adaptation in a foreign host species, i.e.,embryonated chicken eggs, in the following manner. Embryonated, 10 or11-day old chicken eggs, available, for example, from Truslow Farms,Chestertown, Md. or from HyVac, Adel, Iowa, were inoculated with theparental equine influenza virus by injecting about 0.1 milliliter (ml)undiluted AF containing approximately 10⁶ plaque forming units (pfu) ofvirus into the allantoic cavity through a small hole punched in theshell of the egg. The holes in the eggs were sealed with nail polish andthe eggs were incubated in a humidified incubator set at the appropriatetemperature for three days. Following incubation, the eggs were candledand any non-viable eggs were discarded. AF was harvested from viableembryos by aseptically removing a portion of the egg shell, pullingaside the chorioallantoic membrane (CAM) with sterile forceps andremoving the AF with a sterile pipette. The harvested AF was frozenbetween passages. The AF was then used, either undiluted or diluted1:1000 in phosphate-buffered saline (PBS) as noted in Table 1, toinoculate a new set of eggs for a second passage, and so on. A total of69 passages were completed. Earlier passages were done at either about34° C. (passages 1-2) or about 30° C. and on subsequent passages, theincubation temperature was shifted down either to about 28° C., or toabout 26° C. In order to increase the possibility of the selection ofthe desired phenotype of a stable, attenuated virus, the initial serialpassage was expanded to included five different limbs of the serialpassage tree, A through E, as shown in Table 1. TABLE 1 Passage historyof the limbs A through E Passage # Temperature Limb A Limb B Limb C LimbD Limb E 34° C. 1-2  1-2  1-2  1-2  1-2 30° C. 3-8  3-29  3-29  3-29 3-29 28° C. 30-33* 30-68* 30-33 30-69 26° C. 9-65 34-69* 34-65

[0057] B. Virus isolates carried through the cold-adaptation proceduredescribed in section A were tested for temperature sensitivity, i.e., aphenotype in which the cold-adapted virus grows at the lower, orpermissive temperature (e.g., about 34° C.), but no longer forms plaquesat a higher, or non-permissive temperature (e.g., about 37° C. or about39° C.), as follows. At each cold-adaptation passage, the AF was titeredby plaque assay at about 34° C. Periodically, individual plaques fromthe assay were clonally isolated by excision of the plaque area andplacement of the excised agar plug in a 96-well tray containing amonolayer of MDCK cells. The 96-well trays were incubated overnight andthe yield assayed for temperature sensitivity by CPE assay in duplicate96-well trays incubated at about 34° C. and at about 39° C. The percentof the clones that scored as temperature sensitive mutants by thisassay, i.e., the number of viral plaques. that grew at 34° C but did notgrow at 39° C., divided by the total number of plaques, was calculated,and is shown in Table 2. Temperature sensitive isolates were thenevaluated for protein synthesis at the non-permissive temperature byvisualization of radiolabeled virus-synthesized proteins by SDSpolyacrylamide gel electrophoresis (SDS-PAGE). TABLE 2 Percent ofisolated Clones that were temperature sensitive. Percent TemperatureSensitive Passage# Limb A Limb B Limb C Limb D Limb E p36  56%  66%  0% 66% 54% p46  80%  60% 75% p47  80% p48 100% p49 100% 100% 50% p50  90%p51 100% p52 57% p62 100% 100% p65 100% p66 100% 88%

[0058] From the clonal isolates tested for temperature sensitivity, twowere selected for further study. Clone EIV-P821 was selected from the49th passage of limb B and clone EIV-P824 was selected from the 48thpassage of limb C, as defined in Table 1. Both of these virus isolateswere temperature sensitive, with plaque formation of both isolatesinhibited at a temperature of about 39° C. At this temperature, proteinsynthesis was completely inhibited by EIV-P821, but EIV-P824 exhibitednormal levels of protein synthesis. In addition, plaque formation byEIV-P821 was inhibited at a temperature of about 37° C., and at thistemperature, late gene expression was inhibited, i.e., normal levels ofNP protein were synthesized, reduced or no M1 or HA proteins weresynthesized, and enhanced levels of the polymerase proteins weresynthesized. The phenotype observed at 37° C., being typified bydifferential viral protein synthesis, was distinct from the proteinsynthesis phenotype seen at about 39° C., which was typified by theinhibition of synthesis of all viral proteins. Virus EIV-P821 has beendeposited with the American Type Culture Collection (ATCC) underAccession No. ATCC VR-______, and virus EIV-P824 has been deposited withthe ATCC under Accession No. ATCC VR-______.

[0059] C. Further characterization of the mutations in isolate EIV-P821were carried out by reassortment analysis, as follows. Reassortmentanalysis in influenza viruses allows one skilled in the art, undercertain circumstances, to correlate phenotypes of a given virus withputative mutations occurring on certain of the eight RNA segments thatcomprise an influenza A virus genome. This technique is described, forexample, in Palese, et al., ibid. A mixed infection of EIV-P821 and anavian influenza virus, A/mallard/New York/6750/78 was performed asfollows. MDCK cells were co-infected with EIV-P821 at a multiplicity ofinfection (MOI) of 2 pfu/cell and A/mallard/New York/6750/78 at an MOIof either 2, 5, or 10 pfu/cell. The infected cells were incubated at atemperature of about 34° C. The yields of the various co-infections weretitered and individual plaques were isolated at about 34° C., and theresultant clonal isolates were characterized as to whether they wereable to grow at about 39° C. and about 37° C., and express their genes,i.e., synthesize viral proteins, at about 39° C., about 37° C., andabout 34° C. Protein synthesis was evaluated by SDS-PAGE analysis ofradiolabeled infected-cell lysates. The HA, NP and NS-1 proteins of thetwo parent viruses, each of which is encoded by a separate genomesegment, were distinguishable by SDS-PAGE analysis, since theseparticular viral proteins, as derived from either the equine or theavian influenza virus, migrate at different apparent molecular weights.In this way it was possible, at least for the HA, NP, and NS-1 genes, toevaluate whether certain phenotypes of the parent virus. e.g., thetemperature sensitive and the protein synthesis phenotypes, co-segregatewith the genome segments carrying these genes. The results of thereassortment analyses investigating co-segregation of a) the mutationinhibiting plaque formation, i.e., the induction of CPE, at anon-permissive temperature of about 39° C. or b) the mutation inhibitingprotein synthesis at a non-permissive temperature of about 39° C. witheach of the EIV-P821 HA, NP and NS-1 proteins are shown in Tables 3 and4, respectively. TABLE 3 Reassortment analysis of the EIV-P821 39° C.plaque formation phenotype with avian influenza virus, A/mallard/NewYork/6750/78 Gene Virus ts+¹ ts−² HA avian 26 13 equine 11 44 NP avian37 8 equine 0 49 NS-1 avian 9 8 equine 12 20

[0060] TABLE 4 Reassortment analysis of the EIV-P821 39° C. proteinsynthesis phenotype with avian influenza virus, A/mallard/NewYork/6750/78 Gene Virus ts+¹ ts−² HA avian 18 1 equine 11 7 NP avian 345 equine 7 8 NS-1 avian 10 4 equine 14 5

[0061] The results demonstrated an association of the equine NP genewith a mutation causing the inability of EIV-P82 1 to form plaques at anon-permissive temperature of about 39° C., but the results did notsuggest an association of any of the HA, NP, or NS-1 genes with amutation causing the inability of EIV-P821 to express viral proteins ata non-permissive temperature of about 39° C. Thus, these data alsodemonstrated that the plaque formation phenotype and the proteinsynthesis phenotype observed in virus EIV-P821 were the result ofseparate mutations.

[0062] D. Studies were also conducted to determine if cold-adaptedequine influenza viruses of the present invention have a dominantinterference phenotype, that is, whether they dominate in mixedinfection with the wild type parental virus A/Kentucky/1/91 (H3N8). Thedominant interference phenotype of viruses EIV-P821 and EIV-P824 wereevaluated in the following manner. Separate monolayers of MDCK cellswere singly infected with the parental virus A/Kentucky/1/91 (H3N8) atan MOI of 2, singly infected with either cold-adapted virus EIV-P821 orEIV-P824 at an MOI of 2, or simultaneously doubly infected with both theparental virus and one of the cold adapted viruses at an MOI of 2+2, allat a temperature of about 34° C. At 24 hours after infection, the mediafrom the cultures were harvested and the virus yields from the variousinfected cells were measured by duplicate plaque assays performed attemperatures of about 34° C. and about 39° C. This assay took advantageof the fact that cold adapted equine influenza viruses EIV-P821 orEIV-P824 are temperature sensitive and are thus unable to form plaquesat a non-permissive temperature of about 39° C., while the parentalvirus is able to form plaques at both temperatures, thus making itpossible to measure the growth of the parental virus in the presence ofthe cold adapted virus. Specifically, the dominant interference effectof the cold adapted virus on the growth of the parental virus wasquantitated by comparing the virus yield at about 39° C. of the cellssingly infected with parental virus to the yield of the parental virusin doubly infected cells. EIV-P821, in mixed infection, was able toreduce the yield of the parental virus by approximately 200 fold, whileEIV-P824, in mixed infection, reduced the yield of the parental virus byapproximately 3200 fold. This assay therefore showed that cold-adaptedequine influenza viruses EIV-P821 and EIV-P824 both exhibit the dominantinterference phenotype.

[0063] E. Virus isolate EIV-MSV+5 was derived from EIV-P821, as follows.EIV-P821 was passaged once in eggs, as described above, to produce aMaster Seed Virus isolate, denoted herein as EIV-MSVO. EIV-MSV0 was thensubjected to passage three additional times in eggs, the virus isolatesat the end of each passage being designated EIV-MSV+1, EIV-MSV+2, andEIV-MSV+3, respectively. EIV-MSV+3 was then subjected to two additionalpassages in MDCK cells, as follows. MDCK cells were grown in 150 cm²tissue culture flasks in MEM tissue culture medium with Hanks Salts,containing 10% calf serum. The cells were then washed with sterile PBSand the growth medium was replaced with about 8 ml per flask ofinfection medium (tissue culture medium comprising MEM with Hanks Salts,1 μg/ml TPCK trypsin solution, 0.125% bovine serum albumin (BSA), and 10mM HEPES buffer). MDCK cells were inoculated with AF containing virusEIV-MSV+3 (for the first passage in MDCK cells) or virus stock harvestedfrom EIV-MSV+4 (for the second passage in MDCK cells), and the viruseswere allowed to adsorb for 1 hour at about 34° C. The inoculum wasremoved from the cell monolayers, the cells were washed again with PBS,and about 100 ml of infection medium was added per flask. The infectedcells were incubated at about 34° C. for 24 hours. The virus-infectedMDCK cells were harvested by shaking the flasks vigorously to disruptthe cell monolayer, resulting in virus isolates EIV-MSV+4 (the firstpassage in MDCK cells), and EIV-MSV+5 (the second passage in MDCKcells).

[0064] Viruses EIV-MSVO and EIV-MSV+5 were subjected to phenotypicanalysis, as described in section B above, to determine their ability toform plaques and synthesize viral proteins at temperatures of about 34°C., about 37° C., and about 39° C. Both EIV-MSVO and EIV-MSV+5 formedplaques in tissue culture cells at a temperature of about 34° C., andneither virus isolate formed plaques or exhibited detectable viralprotein synthesis at a temperature of about 39° C. Virus EIV-MSVO had asimilar temperature sensitive phenotype as EIV-P821 at a temperature ofabout 37° C., i.e., it was inhibited in plaque formation, and late geneexpression was inhibited. However, EIV-MSV+5, unlike its parent virus,EIV-P821, did form plaques in tissue culture at a temperature of about37° C., and at this temperature, the virus synthesized normal amounts ofall proteins. Virus EIV-MSV+5 has been deposited with the ATCC underAccession No. ATCC VR-______.

EXAMPLE 2

[0065] Therapeutic compositions of the present invention were producedas follows.

[0066] A. A large stock of EIV-P821 was propagated in eggs as follows.About 60 specific pathogen-free embryonated chicken eggs were candledand non-viable eggs were discarded. Stock virus was diluted to about1.0×10⁵ pfu/ml in sterile PBS. Virus was inoculated into the allantoiccavity of the eggs as described in Example 1A. After a 3-day incubationin a humidified chamber at a temperature of about 34° C., AF washarvested from the eggs according to the method described in Example 1A.The harvested AF was mixed with a stabilizer solution, for example A1/A2stabilizer, available from Diamond Animal Health, Des Moines, Iowa, at25% V/V (stabilizer/AF). The harvested AF was batched in a centrifugetube and was clarified by centrifugation for 10 minutes at 1000 rpm inan IEC Centra-7R refrigerated table top centrifuge fitted with aswinging bucket rotor. The clarified fluid was distributed into 1-mlcryovials and was frozen at about −70° C. Virus stocks were titrated onMDCK cells by CPE and plaque assay at about 34° C.

[0067] B. A large stock of EIV-P821 was propagated in MDCK cells asfollows. MDCK cells were grown in 150 cm² tissue culture flasks in MEMtissue culture medium with Hanks Salts, containing 10% calf serum. Thecells were then washed with sterile PBS and the growth medium wasreplaced with about 8 ml per flask of infection medium. The MDCK cellswere inoculated with virus stock at an MOI ranging from about 0.5 pfuper cell to about 0.005 pfu per cell, and the viruses were allowed toadsorb for 1 hour at about 34° C. The inoculum was removed from the cellmonolayers, the cells were washed again with PBS, and about 100 ml ofinfection medium was added per flask. The infected cells were incubatedat about 34° C. for 24 hours. The virus-infected MDCK cells wereharvested by shaking the flasks vigorously to disrupt the cell monolayerand stabilizer solution was added to the flasks at 25% VN(stabilizer/virus solution). The supernatants were distributedaseptically into cryovials and frozen at −70° C.

[0068] C. Therapeutic compositions comprising certain cold-adaptedtemperature sensitive equine influenza viruses of the present inventionwere formulated as follows. Just prior to vaccination procedures, suchas those described in Examples 3-7 below, stock vials of EIV-P821 orEIV-MSV+5 were thawed and were diluted in an excipient comprising eitherwater, PBS, or in MEM tissue culture medium with Hanks Salts, containing0.125% bovine serum albumin (BSA-MEM solution) to the desired dilutionfor administration to animals. The vaccine compositions were held on iceprior to vaccinations. All therapeutic compositions were titered on MDCKcells by standard methods just prior to vaccinations and whereverpossible, an amount of the composition, treated identically to thoseadministered to the animals, was titered after the vaccinations toensure that the virus remained viable during the procedures.

EXAMPLE 3

[0069] A therapeutic composition comprising cold-adapted equineinfluenza virus EIV-P821 was tested for safety and its ability toreplicate in three horses showing detectable prior immunity to equineinfluenza virus as follows. EIV-P821, produced as described in Example1A, was grown in eggs as described in Example 2A and was formulated intoa therapeutic composition comprising 107 pfu EIV-P821/2ml BSA-MEMsolution as described in Example 2C.

[0070] Three ponies having prior detectable hemagglutination inhibition(HAI) titers to equine influenza virus were inoculated with atherapeutic composition comprising EIV-P821 by the following method.Each pony was given a 2-ml dose of EIV-P821, administered intranasallyusing a syringe fitted with a blunt cannula long enough to reach pastthe false nostril, 1 ml per nostril.

[0071] The ponies were observed for approximately 30 minutes immediatelyfollowing and at approximately four hours after vaccination forimmediate type allergic reactions such as sneezing, salivation, laboredor irregular breathing, shaking, anaphylaxis, or fever. The animals werefurther monitored on days 1-11 post-vaccination for delayed typeallergic reactions, such as lethargy or anorexia. None of the threeponies in this study exhibited any allergic reactions from thevaccination.

[0072] The ponies were observed daily, at approximately the same timeeach day, starting two days before vaccination and continuing throughday 11 following vaccination for clinical signs consistent with equineinfluenza. The ponies were observed for nasal discharge, oculardischarge, anorexia, disposition, heart rate, capillary refill time,respiratory rate, dyspnea, coughing, lung sounds, presence of toxic lineon upper gum, and body temperature. In addition submandibular andparietal lymph nodes were palpated and any abnormalities were described.None of the three ponies in this study exhibited any abnormal reactionsor overt clinical signs during the observation period.

[0073] To test for viral shedding in the animals, on days 0 through 11following vaccination, nasopharyngeal swabs were collected from theponies as described in Chambers, et al., 1995, Equine Practice, 17,19-23. Chambers, et al., ibid., is incorporated herein by reference inits entirety. Briefly, two sterile Dacron polyester tipped applicators(available, e.g., from Hardwood Products Co., Guilford, Me.) wereinserted, together, into each nostril of the ponies. The swabs (fourtotal, two for each nostril) were broken off into a 15-ml conicalcentrifuge tube containing 2.5 ml of chilled transport medium comprising5% glycerol, penicillin, streptomycin, neomycin, and gentamycin in PBSat physiological pH. Keeping the samples on wet ice, the swabs wereaseptically wrung out into the medium and the nasopharyngeal sampleswere divided into two aliquots. One aliquot was used to attemptisolation of EIV by inoculation of embryonated eggs, using the methoddescribed in Example 1. The AF of the inoculated eggs was then testedfor its ability to cause hemagglutination, by standard methods,indicating the presence of equine influenza virus in the AF. On days 2and 3 post-vaccination, the other aliquots were tested for virus by theDirectigen® Flu A test, available from Becton-Dickinson (Cockeysville,Md.).

[0074] Attempts to isolate EIV from the nasopharyngeal secretions of thethree animals by egg inoculation were unsuccessful. However on days 2and 3, all animals tested positive for the presence of virus sheddingusing the Directigen Flu A test, consistent with the hypothesis thatEIV-P821 was replicating in the seropositive ponies.

[0075] To test the antibody titers to EIV in the inoculated animalsdescribed in this example, as well as in the animals described inExamples 4-7, blood was collected from the animals prior to vaccinationand on designated days post-vaccination. Serum was isolated and wastreated either with trypsin/periodate or kaolin to block the nonspecificinhibitors of hemagglutination present in normal sera. Serum sampleswere tested for hemagglutination inhibition (HAI) titers against arecent EIV isolate by standard methods, described, for example in the“Supplemental assay method for conducting the hemagglutinationinhibition assay for equine influenza virus antibody” (SAM 124),provided by the U.S.D.A. National Veterinary Services Laboratory under 9CFR 113.2, which is incorporated by reference herein in its entirety.

[0076] The HAI titers of the three ponies are shown in Table 5. As canbe seen, regardless of the initial titer, the serum HAI titers increasedat least four-fold in all three animals after vaccination with EIV-P821.

[0077] These data demonstrate that cold-adapted equine influenza virusEIV-P821 is safe and non-reactogenic in sero-positive ponies, and thatthese animals exhibited an increase in antibody titer to equineinfluenza virus, even though they had prior demonstrable titers. TABLE 5HAI titers of vaccinated animals* Animal HAI Titer (days aftervaccination) ID 0 7 14 21 18 40 80 160 160 19 10 20 40 80 25 20 40 32080

EXAMPLE 4

[0078] This Example discloses an animal study to evaluate the safety andefficacy of a therapeutic composition comprising cold-adapted equineinfluenza virus EIV-P821.

[0079] A therapeutic composition comprising cold-adapted equineinfluenza virus EIV-P821 was tested for attenuation, as well as itsability to protect horses from challenge with virulent equine influenzavirus, as follows. EIV-P821, produced as described in Example 1, wasgrown in eggs as described in Example 2A and was formulated into atherapeutic composition comprising 10⁷ pfu of virus/2 ml water, asdescribed in Example 2C. Eight EIV-seronegative ponies were used in thisstudy. Three of the eight ponies were vaccinated with a 2-ml dosecomprising 10⁷ pfu of the EIV-P821 therapeutic composition, administeredintranasally, using methods similar to those described in Example 3. Onepony was given 10⁷ pfu of the EIV-P821 therapeutic composition,administered orally, by injecting 6 ml of virus into the pharynx, usinga 10-ml syringe which was adapted to create a fine spray by thefollowing method. The protruding “seat” for the attachment of needleswas sealed off using modeling clay and its cap was left in place. About10 holes were punched through the bottom of the syringe, i.e.,surrounding the “seat,” using a 25-gauge needle. The syringe was placedinto the interdental space and the virus was forcefully injected intothe back of the mouth. The remaining four ponies were held asnon-vaccinated controls.

[0080] The vaccinated ponies were observed for approximately 30 minutesimmediately following and at approximately four hours after vaccinationfor immediate type allergic reactions, and the animals were furthermonitored on days 1-11 post-vaccination for delayed type allergicreactions, both as described in Example 3. None of the four vaccinatedponies in this study exhibited any abnormal reactions from thevaccination.

[0081] The ponies were observed daily, at approximately the same timeeach day, starting two days before virus vaccination and continuingthrough day 11 following vaccination for clinical signs, such as thosedescribed in Example 3. None of the four vaccinated ponies in this studyexhibited any clinical signs during the observation period. This resultdemonstrated that cold-adapted equine influenza virus EIV-P821 exhibitsthe phenotype of attenuation.

[0082] To test for viral shedding in the vaccinated animals, on days 0through 11 following vaccination, nasopharyngeal swabs were collectedfrom the ponies as described in Example 3. The nasopharyngeal sampleswere tested for virus in embryonated chicken eggs according to themethod described in Example 3.

[0083] As shown in Table 6, virus was isolated from only one vaccinatedanimal using the egg method. However, as noted in Example 3, the lack ofisolation by this method does not preclude the fact that virusreplication is taking place, since replication may be detected by moresensitive methods, e.g., the Directigen Flu A test. TABLE 6 Virusisolation in eggs after vaccination Animal Virus Isolation (days aftervaccination) ID Route 0 1 2 3 4 5 6 7 8 9 10 11 91 IN −− + + + + + + + + + − 666 IN − − − − − − − − − − − − 673 IN − − − − − −− − − − − − 674 Oral − − − − − − − − − − − −

[0084] To test the antibody titers to equine influenza virus in thevaccinated animals, blood was collected from the animals prior tovaccination and on days 7, 14, 21, and 28 post-vaccination. Serumsamples were isolated and were tested for hemagglutination inhibition(HAI) titers against a recent EIV isolate according to the methodsdescribed in Example 3. TABLE 7 HAI titers after vaccination Animal HAITiter (days after vaccination) ID Route 0 7 14 21 28 91 IN <10 <10 <10<10 <10 666 IN 10 10 10 20 20 673 IN 10 10 10 20 20 674 Oral 20 40 40 4040

[0085] Unlike the increase in HAI titer observed with the three animalsdescribed in the study in Example 3, the animals in this study did notexhibit a significant increase, i.e., greater than four-fold, in HAItiter following vaccination with EIV-P821.

[0086] Approximately four and one-half months after vaccine virusadministration, all 8 ponies, i.e., the four that were vaccinated andthe four non-vaccinated controls, were challenged by the followingmethod. For each animal, 10⁷ pfu of the virulent equine influenza virusstrain A/equine/Kentucky/1/91 (H3N8) was suspended in 5 ml of water. Amask was connected to a nebulizer, and the mask was placed over theanimal's muzzle, including the nostrils. Five (5) ml was nebulized foreach animal, using settings such that it took 5-10 minutes to deliverthe full 5 ml. Clinical observations, as described in Example 3, wereperformed on all animals three days before challenge and daily for 11days after challenge.

[0087] Despite the fact that the vaccinated animals did not exhibitmarked increases in their HAI titers to equine influenza virus, all fourvaccinated animals were protected against equine influenza viruschallenge. None of the vaccinated animals showed overt clinical signs orfever, although one of the animals had a minor wheeze for two days. Onthe other hand, all four non-vaccinated ponies shed virus and developedclinical signs and fever typical of equine influenza virus infection.Thus, this example demonstrates that a therapeutic composition of thepresent invention can protect horses from equine influenza disease.

EXAMPLE 5

[0088] This Example discloses an additional animal study to evaluateattenuation of a therapeutic composition comprising cold-adapted equineinfluenza virus EIV-P821, and its ability to protect vaccinated horsesfrom subsequent challenge with virulent equine influenza virus.Furthermore, this study evaluated the effect of exercise stress on thesafety and efficacy of the therapeutic composition.

[0089] A therapeutic composition comprising cold-adapted equineinfluenza virus EIV-P821 was tested for safety and efficacy in horses,as follows. EIV-P821, produced as described in Example 1, was grown ineggs as described in Example 2A and was formulated into a therapeuticcomposition comprising 10⁷ pfu virus/5 ml water, as described in Example2C. Fifteen ponies were used in this study. The ponies were randomlyassigned to three groups of five animals each, as shown in Table 8,there being two vaccinated groups and one unvaccinated control group.The ponies in group 2 were exercise stressed before vaccination, whilethe ponies in vaccinate group 1 were held in a stall. TABLE 8Vaccination/challenge protocol Group  No. Ponies Exercise VaccineChallenge 1 5 — Day 0 Day 90 2 5 Days −4 to 0 Day 0 Day 90 3 5 — — Day90

[0090] The ponies in group 2 were subjected to exercise stress on atreadmill prior to vaccination, as follows. The ponies were acclimatedto the use of the treadmill by 6 hours of treadmill use at a walk only.The actual exercise stress involved a daily exercise regimen starting 4days before and ending on the day of vaccination (immediately prior tovaccination). The treadmill exercise regimen is shown in Table 9. TABLE9 Exercise regimen for the ponies in Group 2 Speed (m/sec) Time (min.)Incline (°)  1.5 2 0 3.5 2 0 3.5 2 7 4.5† 2 7 5.5† 2 7 6.5† 2 7 7.5† 2 78.5† 2 7 3.5 2 7 1.5 10 0†

[0091] Groups 1 and 2 were given a therapeutic composition comprising10⁷ pfu of EIV-P821, by the nebulization method described for thechallenge described in Example 4. None of the vaccinated ponies in thisstudy exhibited any immediate or delayed allergic reactions from thevaccination.

[0092] The ponies were observed daily, at approximately the same timeeach day, starting two days before vaccination and continuing throughday 11 following vaccination for clinical signs, such as those describedin Example 3. None of the vaccinated ponies in this study exhibited anyovert clinical signs during the observation period.

[0093] To test for viral shedding in the vaccinated animals, beforevaccination and on days 1 through 11 following vaccination,nasopharyngeal swabs were collected from the ponies as described inExample 3. The nasopharyngeal samples were tested for virus inembryonated chicken eggs according to the method described in Example 3.Virus was isolated from the vaccinated animals, i.e., Groups 1 and 2, asshown in Table 10. TABLE 10 Virus isolation after vaccination AnimalVirus Isolation (days after vaccination) Group ID Exercise 0 1 2 3 4 5 67 8 9 10 11 1 12 No − − + + + + + − + + − − 16 − − + + + + + − − − − −17 − − + + + + + + + − + − 165 − − − − − − − − − − − − 688 − − − − − +− + − − − − 2 7 Yes − − − + + + + − − − − − 44 − − − − − − − − − − − −435 − − + + + + − − − − − − 907 − − − + − + + − − − − − 968 − − − − − +− + − − − −

[0094] To test the antibody titers to equine influenza virus in thevaccinated animals, blood was collected prior to vaccination and on days7, 14, 21, and 28 post-vaccination. Serum samples were isolated and weretested for HAI titers against a recent EIV isolate according to themethods described in Example 3. These titers are shown in Table 11.TABLE 11 HAI titers after vaccination and after challenge on day 90Animal Day Post-vaccination Group ID −1 7 14 21 28 91 105 112 119 126 112 <10 <10 <10 <10 <10 <10 80 320 320 640 1 16 <10 <10 20 20 <10 <10 20160 320 320 1 17 <10 <10 10 10 10 10 80 160 160 160 1 165 <10 <10 10 1010 10 80 80 80 80 1 688 <10 <10 20 20 20 20 20 20 20 40 2 7 <10 <10 1010 <10 <10 20 80 80 40 2 44 <10 <10 20 20 20 10 80 320 320 320 2 435 <10<10 20 20 10 <10 20 80 80 80 2 907 <10 <10 10 10 20 10 10 40 80 80 2 968<10 <10 <10 <10 <10 <10 40 160 160 160 3 2 <10 80 640 640 320 3 56 <1080 320 320 320 3 196 <10 20 160 80 80 3 198 10 40 160 320 320 3 200 <1020 80 80 40 Group Description 1 Vaccination only 2 Vaccination andExercise 3 Control

[0095] On day 90 post vaccination, all 15 ponies were challenged with10⁷ pfu of equine influenza virus strain A/equine/Kentucky/1/91 (H3N8)by the nebulizer method as described in Example 4. Clinicalobservations, as described in Example 3, were performed on all animalsthree days before challenge and daily for 11 days after challenge. Therewere no overt clinical signs observed in any of the vaccinated ponies.Four of the five non-vaccinated ponies developed fever and clinicalsigns typical of equine influenza virus infection.

[0096] Thus, this example demonstrates that a therapeutic composition ofthe present invention protects horses against equine influenza disease,even if the animals are stressed prior to vaccination.

EXAMPLE 6

[0097] This Example compared the infectivities of therapeuticcompositions of the present invention grown in eggs and grown in tissueculture cells. From a production standpoint, there is an advantage togrowing therapeutic compositions of the present invention in tissueculture rather than in embryonated chicken eggs. Equine influenza virus,however, does not grow to as high a titer in cells as in eggs. Inaddition, the hemagglutinin of the virus requires an extracellularproteolytic cleavage by trypsin-like proteases for infectivity. Sinceserum contains trypsin inhibitors, virus grown in cell culture must bepropagated in serum-free medium that contains trypsin in order to beinfectious. It is well known by those skilled in the art that suchconditions are less than optimal for the viability of tissue culturecells. In addition, these growth conditions may select for virus withaltered binding affinity for equine cells, which may affect viralinfectivity since the virus needs to bind efficiently to the animal'snasal mucosa to replicate and to stimulate immunity. Thus, the objectiveof the study disclosed in this example was to evaluate whether theinfectivity of therapeutic compositions of the present invention wasadversely affected by growth for multiple passages in in vitro tissueculture.

[0098] EIV-P821, produced as described in Example 1, was grown in eggsas described in Example 2A or in MDCK cells as described in Example 2B.In each instance, the virus was passaged five times. EIV-P821 was testedfor its cold-adaptation and temperature sensitive phenotypes after eachpassage. The egg and cell-passaged virus preparations were formulatedinto therapeutic compositions comprising 10⁷ pfu virus/2ml BSA-MEMsolution, as described in Example 2C, resulting in an egg-grown EIV-P821therapeutic composition and an MDCK cell-grown EIV-P821 therapeuticcomposition, respectively.

[0099] Eight ponies were used in this study. Serum from each of theanimals was tested for HAI titers to equine influenza virus prior to thestudy. The animals were randomly assigned into one of two groups of fourponies each. Group A received the egg-grown EIV-P821 therapeuticcomposition, and Group B received the MDCK-grown EIV-P821 therapeuticcomposition, prepared as described in Example 2B. The therapeuticcompositions were administered intranasally by the method described inExample 3.

[0100] The ponies were observed daily, at approximately the same timeeach day, starting two days before vaccination and continuing throughday 11 following vaccination for allergic reactions or clinical signs asdescribed in Example 3. No allergic reactions or overt clinical signswere observed in any of the animals.

[0101] Nasopharyngeal swabs were collected before vaccination and dailyfor 11 days after vaccination. The presence of virus material in thenasal swabs was determined by the detection of CPE on MDCK cellsinfected as described in Example 1, or by inoculation into eggs andexamination of the ability of the infected AF to cause hemagglutination,as described in Example 3. The material was tested for the presence ofvirus only, and not for titer of virus in the sample. Virus isolationresults are listed in Table 12. Blood was collected and serum samplesfrom days 0, 7, 14, 21 and 28 after vaccination were tested forhemagglutination inhibition antibody titer against a recent isolate. HAItiters are also listed in Table 12. TABLE 12 HAI titers and virusisolation after vaccination HAI Titer (DPV³) Virus Isolation¹ (DPV³)Group² ID 0 7 14 21 28 0 1 2 3 4 5 6 7 8 9 10 11 1 31 <10 20 160 160 160— EC — C EC EC C C EC — — — 37 <10 40 160 160 160 — EC C C EC C C C — —— — 40 <10 20 80 160 80 — EC EC C — C EC C — EC EC — 41 <10 40 160 16080 — EC EC C EC C EC EC — — — — 2 32 <10 <10 80 80 40 — EC — C — C — C —EC — — 34 <10 20 160 160 160 — EC — C EC C EC C — — — — 35 <10 <10 80 8040 — EC — C — C — C — EC — — 42 <10 <10 80 80 40 — — — C — C EC EC — — ——

[0102] The results in Table 12 show that there were no significantdifferences in infectivity or immunogenicity between the egg-grown andMDCK-grown EIV-P821 therapeutic compositions.

EXAMPLE 7

[0103] This example evaluated the minimum dose of a therapeuticcomposition comprising a cold-adapted equine influenza virus required toprotect a horse from equine influenza virus infection.

[0104] The animal studies disclosed in Examples 3-6 indicated that atherapeutic composition of the present invention was efficacious andsafe. In those studies, a dose of 10⁷ pfu, which correlates toapproximately 10⁸ TCID₅₀ units, was used. However, from the standpointsof cost and safety, it is advantageous to use the minimum virus titerthat will protect a horse from disease caused by equine influenza virus.In this study, ponies were vaccinated with four different doses of atherapeutic composition comprising a cold-adapted equine influenza virusto determine the minimum dose which protects a horse against virulentequine influenza virus challenge.

[0105] EIV-P821, produced as described in Example 1A, was passaged andgrown in MDCK cells as described in Example 2B and was formulated into atherapeutic composition comprising either 2×10⁴, 2×10⁵, 2×10⁶, or 2×10⁷TCID₅₀ units/1 ml BSA-MEM solution as described in Example 2C. Nineteenhorses of various ages and breeds were used for this study. The horseswere assigned to four vaccine groups, one group of three horses andthree groups of four horses, and one control group of four horses (seeTable 13). Each of the ponies in the vaccine groupse were given a 1-mldose of the indicated therapeutic composition, administered intranasallyby methods similar to those described in Example 3. TABLE 13 Vaccinationprotocol Vaccine Dose, Group No.  No. Animals TCID₅₀ Units 1 3 2 × 10⁷ 24 2 × 10⁶ 3 4 2 × 10⁵ 4 4 2 × 10⁴ 5 4 control

[0106] The ponies were observed for approximately 30 minutes immediatelyfollowing and at approximately four hours after vaccination forimmediate type reactions, and the animals were further monitored on days1-11 post-vaccination for delayed type reactions, both as described inExample 3. None of the vaccinated ponies in this study exhibited anyabnormal reactions or overt clinical signs from the vaccination.

[0107] Blood for serum analysis was collected 3 days before vaccination,on days 7, 14, 21, and 28 after vaccination, and after challenge on Days35 and 42. Serum samples were tested for HAI titers against a recent EIVisolate according to the methods described in Example 3. These titersare shown in Table 14. Prior to challenge on day 29, 2 of the 3 animalsin group 1, 4 of the 4 animals in group 2, 3 of the 4 animals in group3, and 2 of the 4 animals in group 4 showed at least 4-fold increases inHAI titers after vaccination. In addition, 2 of the 4 control horsesalso exhibited increases in HAI titers. One interpretation for thisresult is that the control horses were exposed to vaccine virustransmitted from the vaccinated horses, since all the horses in thisstudy were housed in the same barn. TABLE 14 HAI titers post-vaccinationand post-challenge, and challenge results Dose in Chall. TCID₅₀ AnimalVaccination on Day 0, Challenge on Day 29 Sick No. units ID −1 7 14 2128 35 42 +/− 1 2 × 10⁷ 41 <10 <10 10 40 10 20 80 − 42 40 40 40 40 40 <1080 − 200 <10 <10 80 40 160 40 40 − 2 2 × 10⁶ 679 <10 10 40 40 40 20 20 −682 <10 <10 40 40 40 40 40 − 795 20 80 160 160 320 320 640 − R <10 10 4020 160 40 40 − 3 2 × 10⁵ 73 <10 <10 160 40 80 160 160 − 712 <10 <10 2020 40 40 20 − 720 <10 20 80 40 80 80 160 − 796 <10 <10 <10 <10 <10 1080 + 4 2 × 10⁴ 75 <10 <10 <10 <10 <10 <10 160 + 724 <10 >10 <10 <10 <1020 320 + 789 <10 10 320 160 320 320 320 − 790 <10 <10 80 40 160 80 40 5Control 12 <10 <10 <10 20 20 40 40 − 22 10 20 40 10 160 40 640 − 71 <10<10 <10 <10 10 20 160 + 74 <10 <10 <10 <10 <10 <10 20 +

[0108] On day 29 post vaccination, all 19 ponies were challenged withequine influenza virus strain A/equine/Kentucky/1/91 (H3N8) by thenebulizer method as described in Example 4. The challenge dose wasprospectively calculated to contain about 10⁸ TCID₅₀ units of challengevirus in a volume of 5 ml for each animal. Clinical observations, asdescribed in Example 3, were monitored beginning two days beforechallenge, the day of challenge, and for 11 days following challenge. Asshown in Table 14, no animals in groups 1 or 2 exhibited clinical signsindicative of equine influenza disease, and only one out of four animalsin group 3 became sick. Two out of four animals in group 4 became sick,and only two of the four control animals became sick. The results inTable 14 suggest a correlation between seroconversion and protectionfrom disease, since, for example, the two control animals showingincreased HAI titers during the vaccination period did not show clinicalsigns of equine influenza disease following challenge. Anotherinterpretation, however, was that the actual titer of the challengevirus may have been less than the calculated amount of 10⁸ TCID₅₀ units,since, based on prior results, this level of challenge should havecaused disease in all the control animals.

[0109] Nonetheless, the levels of seroconversion and the lack ofclinical signs in the groups that received a therapeutic compositioncomprising at least 2×10⁶ TCID₅₀ units of a cold-adapted equineinfluenza virus suggests that this amount was sufficient to protect ahorse against equine influenza disease. Furthermore, a dose of 2×10⁵TCID₅₀ units induced seroconversion and gave clinical protection fromchallenge in 3 out of 4 horses, and thus even this amount may besufficient to confer significant protection in horses against equineinfluenza disease.

[0110] While various embodiments of the present invention have beendescribed in detail, it is apparent that modifications and adaptationsof those embodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

We claim:
 1. A cold-adapted equine influenza virus.
 2. The virus ofclaim 1, wherein said virus replicates in embryonated chicken eggs at atemperature ranging from about 26° C. to about 30° C.
 3. The virus ofclaim 1, wherein said virus is attenuated.
 4. The virus of claim 1,wherein said virus is temperature sensitive.
 5. The virus of claim 1,wherein said virus replicates in embryonated chicken eggs at atemperature ranging from about 26° C. to about 30° C., but does not formplaques in tissue culture cells at a temperature of about 39° C.
 6. Thevirus of claim 1, wherein said virus replicates in embryonated chickeneggs at a temperature ranging from about 26° C. to about 30° C., butdoes not form plaques in tissue culture cells at a temperature of about37° C.
 7. The virus of claim 1, wherein a phenotype comprising anon-permissive temperature of about 39° C. is conferred on said virus byat least two mutations in the genome of said virus, comprising a firstmutation and a second mutation.
 8. The virus of claim 7, wherein saidfirst mutation confers a phenotype on said virus comprising inhibitionof plaque formation at a temperature of about 39° C., and wherein saidfirst mutation co-segregates with the segment of said genome comprisingthe nucleoprotein gene of said virus.
 9. The virus of claim 7, whereinsaid second mutation confers a phenotype on said virus comprisinginhibition of protein synthesis of said virus at a temperature of about39° C.
 10. The virus of claim 7, further comprising at least oneadditional mutation, wherein said additional mutation confers aphenotype comprising a non-permissive temperature of about 37° C. onsaid virus, and wherein said phenotype is selected from the groupconsisting of inhibition of plaque formation at a temperature of about37° C. and inhibition of the expression of the late genes of said virusat a temperature of about 37° C.
 11. The virus of claim 1, wherein saidvirus is produced by a method comprising the steps of: a) passaging awild-type equine influenza virus; and b) selecting viruses that grow ata reduced temperature.
 12. The virus of claim 11, wherein said virus isproduced by a method further comprising repetition of said passaging andselection steps one or more times, wherein said reduced temperature ismade progressively lower.
 13. The virus of claim 11, wherein saidpassaging step is carried out in embryonated chicken eggs.
 14. The virusof claim 11, wherein said virus comprises a dominant interferencephenotype.
 15. The virus of claim 1, wherein said virus is derived fromstrain A/equine/Kentucky/1/91 (H3N8).
 16. The virus of claim 1, whereinsaid virus comprises the identifying phenotypes of a virus selected fromthe group consisting of: EIV-P821, identified by accession No. ATCC VR______; EIV-P824, identified by accession No. ATCC VR ______; and MSV+5,identified by accession No. ATCC VR ______.
 17. The virus of claim 1,wherein said virus is selected from the group consisting of: EIV-P821,identified by accession No. ATCC VR ______; EIV-P824, identified byaccession No. ATCC VR ______; MSV+5, identified by accession No. ATCC VR______; and progeny of any of said viruses having any of said accessionnumbers.
 18. A reassortant influenza A virus comprising at least onegenome segment of an equine influenza virus generated bycold-adaptation, said equine influenza virus having an identifyingphenotype selected from the group consisting of cold-adaptation,temperature sensitivity, dominant interference, and attenuation, whereinsaid equine influenza virus genome segment confers at least one of saididentifying phenotypes to said reassortant virus.
 19. The reassortantinfluenza A virus of claim 18, wherein said virus is produced by amethod comprising the steps of: (a) mixing the genome segments of adonor cold-adapted equine influenza virus with the genome segments of arecipient influenza A virus; and (b) selecting a reassortant viruscomprising at least one phenotype of said donor equine influenza virus,wherein said phenotype is selected from the group consisting ofcold-adaptation, temperature sensitivity, dominant interference, andattenuation.
 20. The reassortant influenza A virus of claim 18, whereinsaid recipient influenza A virus comprises hemagglutinin andneuraminidase phenotypes different than those of said donor equineinfluenza virus, and wherein said reassortant virus comprises thehemagglutinin and neuraminidase phenotypes of said recipient virus. 21.A therapeutic composition to protect an animal against disease caused byan influenza A virus, comprising a virus selected from the groupconsisting of: (a) a cold-adapted equine influenza virus; and (b) areassortant influenza A virus comprising at least one genome segment ofan equine influenza virus generated by cold-adaptation, said equineinfluenza virus having an identifying phenotype selected from the groupconsisting of cold-adaptation, temperature sensitivity, dominantinterference, and attenuation, wherein said equine influenza virusgenome segment confers at least one of said identifying phenotypes tosaid reassortant virus.
 22. The therapeutic composition of claim 21,wherein said therapeutic composition comprises a cold-adapted equineinfluenza virus, wherein said disease is caused by equine influenzavirus, and wherein said therapeutic composition is administeredprophylactically to an equid, thereby eliciting an immune responseagainst equine influenza virus in said equid.
 23. The therapeuticcomposition of claim 21, wherein said therapeutic composition comprisesfrom about 10⁵ TCID₅₀ units to about 10⁸ TCID₅₀ units of said virus. 24.The therapeutic composition of claim 21, further comprising anexcipient.
 25. A method to protect an animal against disease caused byan influenza A virus comprising administering to said animal atherapeutic composition comprising a virus selected from the groupconsisting of: (a) a cold-adapted equine influenza virus; and (b) areassortant influenza A virus comprising at least one genome segment ofan equine influenza virus generated by cold-adaptation, said equineinfluenza virus having an identifying phenotype selected from the groupconsisting of cold-adaptation, temperature sensitivity, dominantinterference, and attenuation, wherein said equine influenza virusgenome segment confers at least one of said identifying phenotypes tosaid reassortant virus.
 26. The method of claim 25, wherein said animalis an equid.
 27. The method of claim 25, wherein said therapeuticcomposition comprises a cold-adapted equine influenza virus, whereinsaid disease is caused by equine influenza virus, and wherein saidtherapeutic composition is administered prophylactically to an equid,thereby eliciting an immune response against equine influenza virus insaid equid.
 28. The method of claim 25, wherein said therapeuticcomposition is administered to said animal by a route that will allowvirus entry into mucosal cells of the upper respiratory tract.
 29. Amethod to produce a cold-adapted equine influenza virus comprising thesteps of: a) passaging a wild-type equine influenza virus; and b)selecting viruses that grow at a reduced temperature.
 30. The method ofclaim 29, wherein said cold-adapted equine influenza virus is producedby a method further comprising repetition of said passaging andselection steps one or more times, wherein said reduced temperature ismade progressively lower.
 31. A method to produce a reassortantinfluenza A virus having at least one genome segment of an equineinfluenza virus generated by cold-adaptation, said equine influenzavirus having an identifying phenotype selected from the group consistingof cold-adaptation, temperature sensitivity, dominant interference, andattenuation, comprising the steps of: (a) mixing the genome segments ofa donor cold-adapted equine influenza virus with the genome segments ofa recipient influenza A virus; and (b) selecting reassortant a viruscomprising at least one phenotype of said donor equine influenza virus,wherein said phenotype is selected from the group consisting ofcold-adaptation, temperature sensitivity, dominant interference, andattenuation.
 32. The method of claim 31, wherein said recipientinfluenza A virus comprises hemagglutinin and neuraminidase phenotypesdifferent than those of said donor equine influenza virus, and whereinsaid reassortant virus comprises the hemagglutinin and neuraminidasephenotypes of said recipient virus.
 33. A method to propagate acold-adapted equine influenza virus comprising a method selected fromthe group consisting of propagating said virus in eggs and propagatingsaid virus in tissue culture cells.